Staphylococcus aureus (SA) has long been recognized as a human pathogen responsible for a wide range of afflictions. Since its first identification in the early 1960s methicillin-resistant Staphylococcus aureus (MRSA) has become one of the most significant pathogens worldwide and is capable of causing a wide range of hospital infections. Staphylococcus aureus infections can be lethal.
Staphylococcus aureus is part of the normal skin flora in healthy persons and is mainly found in the anterior nasal sinus, in the throat, the openings of the mammary glands and on the skin. However, it can cause severe infections when the general condition of the patient is weakened, for example after tissue injury or surgical intervention. SA is described as the most frequent cause of sepsis, skin and soft-tissue infections and pneumonia worldwide.
The ability of SA to adapting to rapidly changing environments was seen in the emergence of Staphylococcus aureus strains that acquired resistance mechanisms to a large number of antimicrobial agents shortly after the introduction of these drugs into clinical practice. Studies have sown that 97% of the Staphylococcus aureus isolates recovered carried resistance to penicillin. The introduction of methicillin in clinical practice was followed by the appearance of the first isolate of Staphylococcus aureus that was resistant not only to penicillin, streptomycin, and tetracycline, but also to methicillin.
The danger from methicillin resistant Staphylococcus aureus (MRSA) as a clinical pathogen is caused by the combination of the resistance gene, which provides resistance to methicillin-type antibiotics, with the particular protective mechanism provided by the enzyme coagulase.
A significant problem in diagnosing Staphylococcus aureus (SA) lies in the differentiation between methicillin resistant, coagulase positive MRSA and methicillin resistant, coagulase negative Staphylococcus (CNS) strains, and in particular in mixtures of such strains with and without resistance. Hence a diagnostic differentiation of CNS from Staphylococcus aureus is needed.
SA strains obtain methicillin resistance through the genomic integration of a gene-fragment, the SSCmec cassette, which in addition to other essential information exhibits the mecA gene, which is responsible for the phenotypical characteristics of resistance.
The SSCmec cassette is integrated at a highly conserved specific site of integration in the chromosomal DNA of SA. This conserved site is positioned close to the 3′ end of an open reading frame (orf X) of the SA genome. It is of significant importance for the present invention that the integration side is highly conserved. However, at least ten various types of the SSCmec cassette with sequence variability have been described in the art.
Another significant problem that arises in mixed cultures of SA and CNS strains, which also may contain strains without the resistance cassette, is that the individual detection of the SA genome and detection of the mecA gene is not sufficient because these gene products may originate from separate individual organisms. Modern molecular diagnostic methods have therefore been developed for the detection of MRSA that preferably detect the junction between the SSCmec cassette and the SA genome.
Examples of such approaches towards detection of the junction between the SSCmec cassette and the SA genome are disclosed in EP 0887424, WO 2002/099034 and WO 2009/085221.
The methods disclosed in these documents relate to polymerase chain reaction (PCR) based methods, which utilize sequence specific primers that specifically hybridize to DNA sequences in the region flanking the cassette-genome junction, so that the target PCR product to be amplified is amplified across the cassette-genome junction.
The methods disclosed in the art do however exhibit significant disadvantages, whereby various false results maybe obtained. Such false results relate commonly to false positives and at times also to false negatives.
The main cause for false positive results is the significant similarity of the junction region of CNS strains with a resistance cassette, in comparison to the analogous junction region of the MRSA strains. Due to this sequence similarity CNS strains with resistance are falsely identified as MRSA. In light of the prior art, novel strategies (for example novel primer sequences) are required in order to avoid false positive identification of CNS. Sequences for primers have until the present time not been developed that enable reliable differentiation between the junction region of CNS strains with a resistance cassette, in comparison to the analogous junction region of the MRSA strains.
False positives may also occur when the junction region is successfully amplified but a deletion of the mecA gene in the integrated cassette has occurred.
The main cause for false negative results is the failure of multiplex primer systems directed towards the various integration sites due to the sequence variation in these regions. The DNA sequence of the SSCmec cassette close to the junction point varies between different MRSA strains. New strains with sequence variation in this region are being discovered regularly, all of which require new primer design and additional forward primers in multiplex reactions, leading to increased failure rates due to multiplex complexity and more expensive amplification reactions. Multiplex design is complicated and labor-intensive, requiring careful sequence selection for compatible melting temperatures of the oligonucleotides applied, whereby multiplex reactions may ultimately need to be avoided for additional security and reliability of the analysis, so that individual reactions should be applied in place of multiplex reactions, thereby leading to increased reagent usage and unwanted associated cost.
For example WO 2009/085221 discloses a method where various forward primers are to be applied within the SSCmec cassette depending on the strain to be interrogated. As can be seen in FIGS. 2 to 4 of WO 2009/085221, multiple forward primers are listed that must be either applied in multiplex or separate reactions, in order to provide sufficient screening of known MRSA strains.
In order to overcome these disadvantages more PCR reactions must be applied (often in complicated multiplex reaction systems) or the sensitivity of the system must be reduced. Neither of these potential solutions represents a satisfactory solution due to increased complexity (and subsequent increased frequency of failure) or lack of reliability.
PCR detection systems for MRSA have been described in the art that exhibit a primer that hybridizes across the junction between the SSCmec cassette and the SA genome (for example the primer is a single DNA oligonucleotide that binds both cassette and genome sequences). Such an approach was described in EP 1529847. However, the oligonucleotide primers described therein exhibit a significant disadvantage, namely, that the melting temperature (Tm) of the primers is approximately between 38 and 48° C., depending on the particular sequence and method used for calculation, resulting in an inability of this method to be effectively applied in practice. Low Tm of the primer causes significant problems in amplification due to the required low annealing temperature (Ta) applied during cycling. In order to enable annealing of a primer with low Tm (such as 48) to its template sequence, a very low Ta must be applied. Due to the low Ta non-specific products tend to amplify in great numbers caused by a high number of base pair mismatches. At low Ta the stringency regarding base pairing is significantly reduced, thereby allowing mismatch annealing and a large number of unspecific products. Mismatch tolerance at low Ta is found to have the one of the strongest influences on PCR specificity.